Effect of physical environment on the conformation of ricin. Influence of low pH.

The molecular properties of ricin (the toxic lectin from Ricinus communis seeds, RCA II or RCA 60) were evaluated by analytical ultracentrifugation, viscosimetry, c.d., fluorescence and equilibrium dialysis. Measurements of sedimentation (S0(20,W) = 4.60 S) and viscosity (eta = 2.96 X 10(-2) dl/g) indicated that, at neutral pH, the ricin molecule is very compact. Various transitions were explored, and a pH-triggered change in the ricin conformation was observed between pH 7 and 4. In this range, the sedimentation coefficient, far-u.v. c.d. and fluorescence altered simultaneously without unfolding. Below pH 7 the change in the ricin conformation was accompanied by a decrease in the affinity of ricin for galactosides, and at pH 4.0 by an alteration in its binding capacity. These effects of low pH are discussed in relation to the physical conditions encountered by ricin molecules during their entry into living cells.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis

1. The pH optima for the incorporation of (14)C-labelled amino acids into gramicidin S by an 11000g cell-free extract from Bacillus brevis have been determined. The pH optima for leucine, proline, phenylalanine, ornithine and valine were 7.5-7.7, 7.5-7.7, 7.7-7.9, 7.7-7.9 and 8.0-8.2 respectively. Hence the greatest difference in pH optima existed between leucine and valine, where it was 0.5pH unit. 2. The 11000g cell-free extract incorporated into gramicidin S only the l-isomers of valine, proline and ornithine. However, both isomers of leucine are utilized and the experiments indicate that a leucine racemase exists in the 11000g cell-free extract. With phenylalanine the l-isomer is utilized much more effectively than the d-isomer. This is noteworthy since it is the d-isomer that occurs in gramicidin S. The experiments indicate that conversion of the l-isomer into the d-form takes place at a stage beyond that of the free amino acid.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

Crystal structure of thermitase and stability of subtilisins

Crystal structure of thermitase, a serine proteinase from Thermoactinomyces vulgaris, has been determined by X-ray diffraction at 1.4 A resolution. The atomic model of thermitase refined to an R-factor of 0.149 contains 1997 protein atoms, 182 water molecules and 2 Ca2+ ions. The tertiary structure of thermitase is similar to that of subtilisin BPN'. The greatest variations are connected with insertions and deletions in the amino acid sequence, which are located on the surface of the molecule. Higher thermostability of thermitase can be explained in terms of the three-dimensional structure. The Ca2+ ions, bound to the protein molecule, as well as the ionic and hydrophobic interactions are supposed to give the main contribution to the stabilization of the structure.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Conformation of 4-thio-l-lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio-l-lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.     

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.